Tyrosine phosphorylation of proteins constitutes a ubiquitous regulatory mechanism which is particularly relevant in multicellular organisms. Protein tyrosine-specific phosphatases (PTPases) likely perform important functions in the regulation of this process. The present proposal considers RPTPalpha, a widely expressed receptor PTPase (R-PTPase), as a model for this class of enzymes. RPTPalpha is implicated in the regulation of Src family kinases, and in the control of cell proliferation and differentiation. The protein is itself tyrosine phosphorylated, and this phosphorylation creates, in vivo, a docking site for the Grb2 protein, a regulator of the Ras/MAP kinase signaling pathway. Three approaches are followed. First, tyrosine phosphorylation of RPTPalpha suggests that this event, and subsequent Grb2 association, constitutes a mechanism which regulates RPTPalpha activity, or/and controls the assembly of a signaling complex including additional proteins. This hypothesis will be tested by kinetic analysis of the effect on enzyme activity, by a biochemical study of the mechanism and affinity of Grb2 association, and through characterization of other proteins which associate with RPTPalpha through its tyrosine phosphorylation site. Second, the function of RPTPalpha will be investigated through expression of wt and mutant RPTPalpha proteins in PC12 cells, an established model for the control of proliferation and differentiation through regulation of the MAP kinase signaling pathway. RPTPalpha expression in these cells has profound consequences on the growth factor specificity of the differentiative response. A study of the mechanisms involved will provide insights into signaling events controlled by RPTPalpha through its catalytic and Grb2 recruitment activities. In addition, RPTPalpha expression will serve as a tool to dissect the specificity of growth factor signaling events in these cells. Finally, an approach is described aimed at identifying new RPTPalpha substrates, through the use of an in vitro binding assay with catalytically inactive RPTPalpha protein. PTPases are increasingly implicated in positive and negative control of cellular signaling events. Therefore, the relevance of this proposal resides in its potential for increased understanding of a class of proteins which may regulate signaling events involved in disease states. Progress made in the study of this particular R-PTPase makes it a particularly promising model system to gain increased understanding of the functions of other members of this broad protein family.